The human liver serves as the reservoir for several important human pathogens, including hepatitis B (HBV) and C viruses (HCV) and Plasmodium species, all of which represent serious global health concerns. HBV and HCV alone chronically infect an estimated 500 million people worldwide, with annual deaths totaling more than 1.5 million. Chronic HBV and HCV infections can have severe health consequences, including hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma. Co-infection with HBV and HCV is common, and leads to a significantly worse prognosis. A preventative vaccine for HBV exists, but curative treatments targeting the virus are not available. Furthermore, increasing HBV resistance has been reported to first-line antiviral drugs. A vaccine for HCV has not yet been developed, and, while HCV-specific protease and polymerase inhibitors are showing promise in early clinical development, rapid emergence of resistance indicates that additional targets and combinations of antivirals will be needed for effective control. The scarcity of in vitro and in vivo systems that faithfully mimic liver biology and susceptibility to human hepatotropic pathogens has severely hampered drug and vaccine development. Here, we propose to take an interdisciplinary approach and combine tissue engineering with molecular virology and humanized mouse technology to create platforms that will facilitate studies of basic virus-host and virus-virus interactions, promote understanding of the mechanisms of liver disease progression, and provide predictive systems to test drug and vaccine efficacy and toxicity. Specifically, we aim to characterize HBV and HCV biology and model associated liver disease in micropatterned primary human hepatocyte cultures (MPCCs) - a breakthrough technology that was recently developed in Dr. Sangeeta Bhatia's laboratory. We will extend this methodology to develop three-dimensional liver organoids, and investigate HBV and HCV infection in mice transplanted with these structures. Since chronic inflammation plays a significant role in liver disease progression, we aim to incorporate components of the immune system in static and dynamic tissue culture systems and ultimately to use mice reconstituted with a human hematopoietic system recipients for HBV/HCV-permissive liver organoids. Through the development and use of these platforms, we hope to shed light on HBV and HCV virology and pathogenesis, and to uncover novel avenues for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Hepatitis B and C viruses are leading causes of human liver disease including cancer. This proposal aims to create and utilize novel primary human hepatocyte tissue culture systems and complex 3D liver organoids to study HCV and HBV mono- and co-infections. The resulting findings should aid development of more effective treatments aimed at eradicating these deadly viral diseases.